This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: The first step in beginning a tomographic study is to clarify precisely what the investigator hopes to learn from a reconstruction. We may find that a simple tilt series animation, or the use of stereopair images with Sterecon is more appropriate. Once we agree that a tomographic study is appropriate, the choice of specimen thickness and magnification have to be carefully considered. The user is then trained in recording the tilt series with the appropriate electron microscope. In some cases, our staff does the microscopy. Protection of the specimen from contamination and excessive irradiation are important. If images are recorded on film, we assist the user in setting up the scanning camera for image digitization and pre alignment. The final alignment normally uses gold markers as fiducial marks and is done using WEB, then the reconstruction is computed using SPIDER. Visualization of the reconstruction is the most time consuming portion of the project. In many cases, we do this with the investigator looking on. If the investigator is to do this independently, there must be training in the use of SPIDER and various visualization packages. Some investigators have visualization capabilities in their own labs, and if they use software we are familiar with (or supply), we also provide assistance, usually over the Internet. Full documentation on the tomographic reconstruction methods we use is available on the SPIDER homepage, and is updated periodically.